Localization and phosphorylation of nuclear, nucleolar and extranucleolar non-histone proteins of Novikoff hepatoma ascites cells

J. Mol. Biol. (1975) 97, 611-619

Localization and Phosphorylation of Nuclear, Nucleolar and Extranucleolar Non-histone Proteins of Novikoff Hepatoma Ascites Cells MARK O. J. 0LSON, EDWARD G. EZRAILSON,KARL GUETZOWAND HARRIS BUSCH

Nuclear Protein Laboratory DeTartment of Pharmacology Baylor College of Medicine Houston, Texas 77025, U.S.A. (Received 13 January 1975, and in revised form d June 1975) The 32p labeling and localization of non-hlstone proteins in chromatin were studied in Novikoff hcpatoma ascites cells i~, ~vo and in vi~e. Chromatin was prepared from whole nuclei, nucleolar and extranucleolar fractions by successive washes with 0.075 ~.NaC1, 0.025 ~-EDTA, 10 m~-NaHSOa, 10 m~-KF, 0"1 m~. phenylmethylsulfonylfluoride(pH 8"0), and with 5 mM-Trls, 5 mM-K.F (pH 8"0). After removal of histones by 0.4 N-H2SO4 and digestion of the DNA by I)Nase I, the non-histone proteins were subjected to two-dimensional electrophoresis and autoradiography. Although most non-histone proteins were common to both fractions, the nucleolar fraction was enriched in proteins C18, C21, Cg' and CB (Yeoman etal., 1973). Proteins CiVI,C6, CA, Cb, B24, B22 and BF were found in higher concentrations in the extranucleolar component. In the nucteolar fraction, only protein C18 was labeled significantly with s2p. In the extranucleolar fraction, a2p was incorporated into protein spots C18, Cg', CM, CN and C6. Analyses of 32P-labeled spots for phosphoamlno acids indicated that all spots analyzed contained 8~P-labeled phosphoserine. These data indicate the nucleolus specific localization of some 32P.labeled non-histone chromatin proteins. 1. I n t r o d u c t i o n Covalently ]inked phosphoryl groups are found in virtually all classes of nuclear proteins. Phosphory]ation of histones has been implicated in the control of gene expression (Langan, 1969), celt division (Balhorn et ~., 1971,1972) and histone removal during spermiogenesis (Marushige ~ al., 1969). Non-histone phosphoproteins specifically bind to (Teng et al., 1971; Kleinsmith, 1973), and stimulate the template activity of DNA (Teng e~ al., 1971). Their phosphorylation is modified by mitogenie agents (Kleinsmith etal., 1966; Johnson etal., 1974) and azo dyes (Chin e~ aL, 1973); it is dependent on the phase of the cell cycle (Karn e~ al., 1974). Recently, numerous phosphoproteins were found in acid extracts of nucleoli (Olson etal., 1974a) and nucleo]ar preribosomal particles (01son et at., 1974b) of Novikoffhepatoma aseites cells. Enhanced levels of transcription and translation generally require a concomitant increase in the synthesis of ribosomes by the nucleolus (Smetana & Busch, 1974). Consequently, some of the changes in the content or phosphorylation of non-histone proteins under conditions of gene activation may be attributed to changes in the 611

612

M.O.J.

OLSON E T A L .

nucleolar component of ehromatin. Some studies have been done to compare the proteins associated with nueleoIar and extranueleolar chromatin (Wilhelm et al., 1972; Gm~mmt, 1974) but previously, characterization of nucleolar specific nonhistone chromatin proteins was reported only for Physarur~ Tolycephalu~ (LeStourgeon & Rusch, 1973). This study was initiated to determiue whether, in Novikoff hepatoma aseites cells, (1) any non-histone proteins are localized in either the nucleolar or extranucleolar chromatin fraction, and (2) a n y of the proteins in either fraction are specifically labeled with s2p.

2. Materials and M e t h o d s (a) AnirnaZ8 and t~zmor c~ls Novikoff bepatom~ ascites cells were transplanted 6 days prior to carrying out experiments in male albino rats obtained from the Holtzma~ Co., Madison, Wisconsin. The chromatin proteins were labeled by intraperitoneal injection of 20 mCi per animal of carrier-frce [32P]orthophosphate (neutralized with 1 N-NaOH and diluted with 0.155 MNaC1) 2 h before the animals were killed. The Novikoff hepatoma ascites cells were drained from the abdominal cavity, filtered through cheesecloth and washed several times with Na]K/Mg buffer (0.13 ~-NaC1, 0.005 ~-KC1, 0.008 ~-MgC12). In some experiments, Novikoff hepatoma ascites cells were labeled for 2 or 18 h in suspensions (Mauritzen et al., 1970) with incubation mixtures containing [82P]orthophosphate (100 mCi/30 g of cells). (b) Preparation of chroraatin Nuclei were prepared by the citric acid method as described by Taylor et al. (1973). Whole nuclear chromatin was prepared by washing nuclei 3 times with 10 vol. 0-025 ~EDTA, 0.075 ~-NaCI, 5 m~t.Na~S03, 10 m~-KF, 0.1 mM-phenylmethysulfonylfluoride (pH 8.0), and 3 times with 10 vol. 5 m~-Tris.HC1, 5 m~-KF (pH 8.0), according to the method of Marushige & Bonner (1966) as modified by Yeoman et al. (1973). For the separation of nucleolar and extranucleolar chromatin, nuclei were suspended in 0.34 ~sucrose and disrupted by sonication until no nuclei remained intact (Busch & Smetana, 1970). The sonieated nuclei were layered over 0.88 M-sucrose and centrifuged at 1100 g for 15 m~n. The pellet (nucleoli) was washed with satine/EDTA and Tris, as above, to prepare nucleolar chromatin. The supernatant solution (extranucleolar material) was made to 0.025 ~-EDTA, 0.075 M.NaC1, 5 n~-NaHS0~, 1 mM-phenylmethylsulfonylfluoride (pH 8.0), and centrifuged at 50,000 g for 90 min. The pellet from this centrifugation was washed successively as described above to prepare extranucleolar chromatin. (e) Ext~raction of proteins The chromatin preparations were extracted twice with 10 vol. 0.4 N-H2SO4 at 4°C by stirring for 4 h. The proteins from the combined acid extracts were designated "chromatin fraction r ' . After extraction of the chromatin preparations with acid, the pellets were homogenized in 2 rnM-CaCI~, 2 m~-]YIg~, 0.1 •-Trls-HCl (pH 7-5) at a concentration of 2 mg/ml. The I)NA was digested according to the method of Wilson & Spelsberg (1973) by addition of 25 ~g deoxyribonuclease I (Worthington Biochemicals, Freehold, N. J.) per mg of pellet at 37°C for 30 rain. The proteins were precipitated by addition of perchloric acid to a concentration of 0.4 z~t. The precipitated proteins were suspended in t In some samples the proteins were washed at this stage with 10 vol. chloroform/methanol (2:1, v/v) containing 0.1 ~-HCI, followed by 10 vol. chloroform/methanol (1:1, v/v) containing 0.1 z¢-HCIand finally with ether. Since omission of this procedure did not alter the autoradiographio patterns, it was not included in most exper/mente.

NOlq-HISTONE PHOSPHOPROTEINS

613

0-9 Y-acetic acid, 10 ~-urea, 1% ~-mereaptoethanol a n d dialyzed aga~_st 2 changes o f 500 vol. of t h e same solution. The proteins soluble after this t r e a t m e n t were designated " c h r o m a t i n fraction I I " . (d) One- and two-dlmensional gel eleoOrophorenis Proteins of chromatin fraction I I were concentrated to 10 mg/ml a n d applied to the modified 2-dimensional s y s t e m described b y Busch e t a / . (1974) using gels containing 6 % acrylamide in the first dimension a n d 8 % acrylamide in the second dimension. A f t e r electrophoresis, t h e 2-dimensional gels were strained with Coomassie brilliant blue R (Sigma, St. Louis, Me.) a n d destained as previously described (Orrick eta/., 1973). Some of t h e first-dimensional gels were stained for 1 h with 1% Buffalo black (Eastman, Rochester, N.Y.) and destained with 7 % acetic acid, 10% methanol prior to slicing for analysis of radioactivity.

(e) Detection of 82P-labeled protein~ A f t e r 24 to 48 h of dest~ining, the slab gels were dried in a B i o R a d model SE540 gel drier (BioRad Laboratories, Richmond, Calif.), w r a p p e d in Saran w r a p so t h a t one side of t h e gel was covered b y only one layer of t h e Saran wrap. This side of t h e gel was t a p e d to a shoot of K o d a k R P R o y a l X - c r o a t X - r a y film (Kodak, Rochester, N.Y.) which was exposed in the d a r k from 1 to 14 days. After development, the stained spots were m a t c h e d with spots on the film. One-dimensional gels were scanned for stained protein bands a t 540 n m in a Gilford model 2000 speetrophotomoter equipped w i t h a linear t r a n s p o r t unit (Gifford Instruments, Oberlin, Ohio}. The gels were then frozen in solid CO2/acetone a n d cut into l - r a m slices with a B i o R a d model 190 gel slicer (BioRad Laboratories, Richmond, Calif.). The slices were placed in scintillation vials a n d dried overnight. After addition of 8 ml Aquasol (New E n g l a n d Nuclear, Boston, Mass.) to each vial, t h e samples were counted in a B e c k m a n LS230 scintillation counter (Beckman I n s t r u m e n t s , P a l e Alto, Calif.). (f) Determination of phosphoamino acids The presence of a2p in phosphoserine a n d phosphothreonine was determined after hydrolysis of t h e individual proteins excised from the gels. Stained spots were cut out of the 2-dimensional gels, dried in vacuo a n d t h e n h y d r o l y z e d in 1 ml 2 N-HC1 a t 110°C for 8 h in vacuo. The hydrolysates were analyzed for 32p incorporated into phosphoserine a n d phosphothreonine on columns of Dowex 50W.X8 as previously described b y Olson etal. (1974a). (g) De~wmina~ion of D N A , protein and R N A Samples from nueleolar a n d extranucleolar fractions were exhaustively dialyzed against 5 m M - N a 0 H a t 4°0 a n d subsequently subjected to analysis for DNA, protein mad R N A . Protein was determined b y the m e t h o d of L o w r y eta/. (1951). D N A was determined b y t h e m e t h o d of R i c h a r d s (1974) after hydrolysis o f the samples with 1.4 1¢-HC104 a t 70"C for 30 rain a n d t h e removal of precipitated protein b y centrifugation. F o r R N A d e t e r m i n a t i o n t h e sample was h y d r o l y z e d with 0-3 ~ - K O H a t 37°C for 18 h, acidified w i t h a n equal vol. o f cold 0-5 N-HCIO~, a n d centrifuged to remove t h e precipitate. The R N A contents of the s u p e r n a t a n t solutions were analyzed b y the orcinol m e t h o d (Drury, 1948). (h) D6terralnation of protein molecular weight8 S t a n d a r d proteins (bovine albumin, ovalbumln a n d chymotrypsinogen) were a p p l i e d to first-dlmensional gels along with a cytochrome c marker. Electrophoresis was cont i n u e d until t h e cytochrome c m a r k e r was 1 c m into t h e gels. T h e gels were a d a p t e d as a b o v e a n d 1.em sections o f t h e gels containing t h e s t a n d a r d proteins were applied along with non-histone protein-containing gels to t h e second dimension slab a t t h e lower melee cular weight side of the sample gels. Molecular weights of representative protein spots (Plate I(a)) were calculated from s t a n d a r d curves as described b y Shapiro et al. (1967),

614

M. O. J. OLS01~ E T A L . 3. R e s u l t s

(a) Characterization of proteins in ~hromatin fractions :Electrophoresis in the two-dimensional system separated the non-histone proteins from whole nuclear, nucleolar and extranucleelar ehromatin (Plate ][(a) to (c)) into approximately 50 spots. The pattern was divided into the B and C regions and the spots were numbered according to the system of Yeoman et al. (1973). Although most of the proteins were present in all three preparations, some proteins were distinctly concentrated in either the nucleolar or extranucleolar fractions (Table 1). For example, the nucleolar fraction was enriched in spots C21, C18 and Cg'. These spots are greatly reduced in intensity in the extranueleolar portion. On the other hand, the extranucleolar chromatin contains higher concentrations of spots BC, B22, B24, Bj, BF and CA. The whole nuclear pattern appears to be a composite of the spots in the extranucleolar and nueleolar chromatin fractions. TABLE 1

Distribution o] Troteins in nucleolar and extranucleolar chromatin Proteins increased in nucleolar fraction

Proteins increased in extranucleolar fraction

C21t, C18t, Cg't

BC, B22, B24, C6~, Bj, BF, CA

J""q~P-labeledin nucleolar chromatin fraetion II. s2p-labeled in extranucleolar chromatin fraction II. (b) Distribution of asp label in the c h r o ~ i n fraction 1"I proteins After two-dimensional gel eleetrophoresis, the 82P-labeled chromatin fraction H proteins were detected by autoradiography. The whole chromatin pattern was similar to the extranueleolar profile, i.e. spots C18, CQ, CT, CT', CW, CZ, CZ', Cm, Cg', CTI, CN, C6 and CC were labeled. In the autoradiograph of the nucleolar fraction, the 32p was concentrated primarily in spot C18. The adjacent spots Cg' and C21 were much less labeled as were spots C1 and C6. The 32p in spot C1 appears to be concen. trated only in the nucleolar fraction. For further quantitation of the 82p uptake into these proteins, one-dimensional gels were sliced and counted in the scintillation counter (Fig. 1). The uptake of 82p is generally distributed throughout the gels with a prominent peak of radioactivit~ around slice 57 in both the whole chromatin and nucleolar chromatin fractions (Fig. l(a) and (b), respectively). This peak corresponds to the position of spot C18 in the two-dimensional gel pattern (Plates I and II) and contains less 32p in the extranueleolar fraction (Fig. 1(c)). (c) Determination of 82p incorporated into phosphoamino acids The proportion of s2p incorporated into proteins in the form of phosphoserine and phosphothreonine was determined by analyses of hydrolyzed proteins in the various gel spots (Olson et al., 1974a). The results for the major labeled spots are summarized

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PLATE I. Two-dimensional polyacrylamide gel electrophoretie p a t t e r n s of 250/zg of c h r o m a t i n fraction r [ proteins. Samples were r u n in the first dimension (horizontal arrow) on disc gels of 6 % polyaerylamide, 6 M-urea, 0.9 :~T-acctie acid a t 120 V for 6 h. For the second dimension (vertical arrow), a n 8 % polyacrylamide, 0"1% sodium dodecyl sulfate slab gel was r u n for I4 h a t 50 mA]slab. Gels were stained with Coomassie brilliant blue R. O indicates origin of sample. Numbers a t right of Plate I(a) indicate molecular weights in thousands as d e ~ r m i n e d b y molecular weight s t a n d a r d proteins (bovine albumin, o v a l b u m i n a n d chymotrypsinogen). (a) Whole nuclear ctn.omatin fraction I I proteins. (b) t~Tucleolar c h r o m a t i n fraction I I proteins. (c) Exgranucleolar c h r o m a t i n fraction I I proteins. [facing p. 614

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PLA2E II. Autoradiograms on X - o m a t X - r a y film of a2P-labeled e h r o m a t i n fraction I I proteins subjected to two-dimensional electrophoresis as described in ~he legend to Plato I. Spots on autoradiograms were m a t c h e d with stained spots a n d n u m b e r e d as in Plato I. lqlu-nbers followed b y P indicate radioactive spots t h a t do not co-migrate with stained spots. (a) Whole nuclear c h r o m a t i n fraction I I proteins. (b) Nucleolar c h r o m a t i n fraction I I proteins. (c) Extranucleolar c h r o m a t i n fraction I I proteins.

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FIG. 1. One-r]Jmensional polyaorylamide gel eleotrophoresJs of a~P-labeled ohromatin fraction I I proteins. The proteins (100 ~g) were subjected to eleotrophoresis as described for the first dimension of the two-dimensional system (Plate I). After staining ~rith Buffalo black, the gels were destained and out into l-ram slices. After drying the slices in scintillation vials, 8 ml Aquasol wore added and the slices were counted in the scintillation counter. Arrow indicates direction of migration. (a) Nuclear ohromatin phosphoproteins; (b) nuoleolar chromatin phosphopro~e~us~ {oI e xt..r~o nuoleolar ehroma~in phosphoproteins.

M. O. J . O L S O N E T AL.

616

TABI~ 2 Recovery of ~2p in larotein spots as lahosphoamino acids

Spot no.

% of total cts/min in spot as Phosphoserine t Phosphothroonine$

Nueleolar C1 C18 C6

82 99 83

7 4 5

Extranueleolar C6 Cg" CN

83 93 83

6 10 11

Analyses of stained spots or a2P-labeled spots for saP-labeled phosphoserine and phosphothreonine. Stained spots corresponding to the numbers in Plates I and I I that contained more than 100 ets]mln per spot were cut out and hydrolyzed with 2 N-HC1 at ll0°C for 8 h in vacuo. After hydrolysis, extracts of the gel plugs were applied to Dowex 50-X8 column~ (0"7 em × 9 cm) and eluted with 0.05 ~r.HC1 as described in text. Unlabeled carrier phospheserine and phosphothreonine were added with the samples to identify the radioactive peaks by uiuhydrin color. t Corrected for 70% destruction during hydrolysis. ~; Corrected for 20% destruction during hydrolysis. i n T a b l e 2. P h o s p h o s e r i n e a n d t r a c e s o f p h o s p h o t h r e o n i n e were f o u n d i n all o f t h e s p o t s a n a l y z e d . I n m o s t cases, m o r e t h a n 70~/o o f t h e t o t a l r a d i o a c t i v i t y was r e c o v e r e d a s p h o s p h o s e r i n e . A c c o r d i n g l y , m o s t o f t h e labeling d e t e c t e d o n t h e t w o - d l m e n s i o n a l gels is f r o m p h o s p h o r y l a t e d p r o t e i n s . (d) Distribution of DNA, protein, RIVA and asp label in the chromatin fractions T h e chemicM c o m p o s i t i o n s o f t h e c h r o m a t i n f r a c t i o n s a r e s h o w n i n T a b l e 3 for a t y p i c a l e x p e r i m e n t . T h e p r o t e i n t o I ) N A r a t i o s were a p p r o x i m a t e l y 2 : 1 for b o t h fractions. S m a l l a m o u n t s o f R N A were also p r e s e n t i n t h e w a s h e d c h r o m a t i n p r e p a r ations. TABLE 3

Distribution of DNA, protein R N A and radioactivity in chromatin fractions Chromatin fraction Nuoleolar Extranueleolar DNA (rag) Protein (mg) Protein~DNA R N A (rag)

a=p (ors/rain X 10 -e)

7-0 14-6 2-1

13.4 26.8 2-0

3-0

2.4

32-2

52-7

Determ~,~ation of DNA, RNA, proteins and ots/r-~u in fractions of chromatin. Samples of the nuolsolar and extranueleolar fractions were analyzed, as desoribed in the text, before and after the washing procedures.

NON-HISTONE PHOSPHOPROTEINS

617

4. Discussion This s~udy indicates that nucleolar chromatin contains specific proteins which have a unique 32p labeling pattern. A number of studies have been reported in which differences were found in the ehromatin non-histone proteins and their phosphorylation in various tissues (Yeoman e~ al., 1975; Teng et al., 1971; MacGinivray ~ ~., 1972; MacGill/vray & Rickwood, 1974), or in the same tissue under different physiological conditions (Johnson et al., 1974; Shelton & A]]frey, 1970; Levy d al., 1973), or in various stages of the cell cycle (Karn et al., 1974; Morales et al., 1974). With the identification of nucleolar-specific non-histone protein, it should be possible to identify the changes in the total non-histone protein due to changes in the activity of the nucleolus. A recent study by Yeoman ~ al. (1974) indicated that a number of non-histone proteins were found in higher concentrations in growing tissues than in non-growing tissues. Among these were C18, CQ and C21, which also appear to be concentrated in the nucleolar component of chromatin. Therefore, it is likely that many of the changes observed in the non-histone protein of whole chromatin during gene activation may reflect nucleolar activity. This type of change in non-histone protein is similar to that due to increased amounts of heterogeneous nuclear ribonuclear particles in chromatin after cortisol stimulation in rat lever (Pederson, 1974). The differences in the proteins of nucleolar and extranucleolar chromatin are not likely to be due to selective contamination of either fraction by cytoplasmic proteins. The citric acid procedure (Taylor e~ al., 1973) removes almost all the cytoplasmic tags as well as the outer layer of the nuclear envelope. Contamination by membrane in either fraction was m~nlmal as seen by electron microscopy (Daskal, personal communication). Although proteins are extracted from nuclei during the citric acid procedure, none of the specifically localized proteins are extracted, as indicated by extraction with 0"5~/o citric acid of nuclei isolated in sucrose (Taylor e~ al., 1973). Furthermore, examination of the two-dimensional gel patterns of acid-soluble proteins from whole nucleoli indicated that the patterns obtained from nucleoli prepared from sucrose-isolated nuclei were essentially identical to those obtained from nucleoli prepared using the citric acid procedure. Therefore, the specific localization of proteins is not the result of the extraction procedure. The method of chromatin fractionation used here must be considered as enrichment rather than a clear-cut separation. Isolation of nucleoli requires extensive sonication to break all nuclei and free the nucleoli of attached chr0matin. In this process, some nucleoli are broken, resulting in contamination of the extranucleolar fraction with small amounts of nucleolar products. For this reason, small amounts of proteins which appear to be nucleolus-specific are found in the extranucleolar fractiont. By several criteria, the 32p uptake was shown to be primarily into proteins. All spots that were analyzed contained phosphoamlno acids (Table 2). Further, washing the proteins with lipid solvents did not alter the autoradiographic pattern, thereby eliminating contamination by membrane phospholipid. In addition, two-dimensional A portion (about 30%) of the extranucleolar ehromatin whioh is not pelleted at 50,000 g in the initial spin can be pelletedby sedimentationat 300,000 g for 16 h. This material is qualitatively and quantitatively s~m~l~rto the 50,000 g pellet in protein content and 8~p labelingbut cont~n~ some fregments of broken nueleoli and lower molecular weight ehromatin resulting from the sonication procedure.

618

M. O. J. OLSON E T A L .

gels containing 32P-labeled proteins were heated in 5~/o trichloroacetic acid at 94°C for 15 minutes. The gels were rinsed twice in cold trichloroacetic acid and autoradiographed. No change was noted in the autoradiographic pattern, indicating t h a t contamination b y RNA was negligible. Although radioactivity was concentrated in a few well-defined spots, 32p was distributed throughout the patterns. In addition, some radioactive spots did not migrate with stained spots (e.g. CBP). This phenomenon was observed by K a r n et al. (1974) in HeLa cell nuclear non-histone protein. I t is possible t h a t some proteins that are present in quantities too small to detect by staining incorporate 82p and have high specific activities. These studies do not permit the assignment of function to nucleolus-specific proteins nor do they indicate that they are associated with specific genes. As isolated, the nucleoli contain virtually all the ribosomal eistrons (Sitz et al., 1973). However, they account for less than 1 ~ of the I)NA of the Novikoff hepatema nucleolus. I t remains to be determined which, ff any, of these proteins are specifically localized to ribosomal I)NA. The nucleolus also contains large amounts of heterochromatin (Busch & Smetana, 1970) which also m a y be associated with specific proteins. The work was supported by a Cancer Research Center grant (no. CA-10893, p.3), an American Cancer Society Institutional Research grant, the Welch Foundation and a gift from Mrs Jack Hutchins. One of us (E. G. E.) was a postdoctoral trainee of the National Cancer Institution CA05154. REFERENCES Balhorn, 1%., 1%ieke, W. O. & Chalkley, 1%. (1971). Biochemist,'y, 19, 3952-3959. Balhorn, 1%., Ch,.]bley, 1%. & Granner, D. (1972). Biochemistxy, 11, 1094-1098. Busch, H. & Smetana, K. (1970). The lVucleolus, Academic Press, New York. Busch, G. I., Yeoman, L. C., Taylor, C. W. & Busch, H. (1974). Phy~dot. Chem. & P h y s i c , 6, 1-10. Chiu, J.-F., Craddock, C., Getz, S. & Hn~]iea, L. S. (1973). F E B S LeUers, 33, 247-250. Drury, H. F. (1948). Arch. Bioehem. 19, 455-466. Gin,mint, I. (1974). F E B S Le~ers, 39, 125-128. Johnson, E. M., Karn, J. & Allfrey, V. G. (1974). J . Biol. Chem. 249, 4990-4999. Karn, J., Johnson, E. M., Vidali, G. & Anfrey, V. G. (1974). J . Biol. Chem. 249, 667-677. Kleinsmith, L. J. (1973). J . Biol. Chem. 248, 5648-5653. Kleinsmith, L. J., Ai1~ey, V. G. & 1Kirsky, A. E. (1966). Proc. N a t Aead. Sci., U.S.A. 55, 1182-1189. Langan, T. A. (1969). Proc. 2~a~. Acad. Sci., U.S.A. 64, 1276-1283. LeStourgeon, W. M. & 1%usch, H. P. (1973). Arch. Bioehem. Biophys. 155, 144-158. Levy, 1%.,Levy, S., 1%osenberg, S. A. & Simpson, 1%. T. (1973). Biochemistry, 12, 224-228. Lowry, O. It., 1%osebrough, N. J., Farr, A. L. & Randall, 1%. J. (1951). J . Biol. Chem. 193, 265-275. MacGillivray, A. J. & 1%ickwood, D. (1974). Eur. J . Bioehem. 41, 181-190. MacGillivray, A. J., Cameron, A., Krauze, 1%. J., 1%ickwood, D., Paul, U. J. (1972). Bioehim. Biophys. Aeta, 277, 38~ ~02. Marushige, K. & Bonner, J. (1966). J . Mol. Biol. 15, 160-174. Marushige, K., Ling, V. & Dixon, G. H. (1969). J . Biol. Chem. 244, 5953-5958. Mauritzen, C. tVI.,Choi, Y. C. & Busch, H. (1970). InMethods inCaneer Research (Busch, H., ed.), vol. 6, pp. 253-282, Academic Press, New York. Morales, M. M. de, Blat, C. & Harel, L. (1974). Exptl. Cell Res. 86, 111-119. Olson, M. O. J., Orrick, L. 1%.,Jones, C. & Busch, H. (1974a). J. Biol. Chem. 249, 2823-2827. Olson, M. O. J., Prestayko, A. W., Jones, C. E. & Busch, H. (1974b). J . Mot. Biol. 90, 161-168.

NON-HISTONE PHOSPHOPROTEINS

619

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